Isolation and characterization of metalloproteases with a novel domain structure by construction and screening of metagenomic libraries

Abstract

Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per microg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65 degrees C, respectively, when casein was used as substrate.

FIG. 1.

Comparison of the inserts of pTW2 (A) and pTW3 (B) with correlating genomic regions of X. campestris pv. vesicatoria strain 85-10 (C), X. campestris pv. campestris strain ATCC 33913 (D), X. campestris pv. campestris strain 8004 (E), X. axonopodis pv. citri strain 306 (F), X. oryzae pv. oryzae KACC 10331 (G), and X. oryzae pv. oryzae MAFF 311018 (H). Arrows and arrowheads indicate the lengths, location, and orientations of potential genes. Genes sharing the same predicted functions are marked with identical colors. Gray arrows and open arrows indicate conserved hypothetical proteins and hypothetical proteins, respectively. Genes encoding putative proteases are boxed in yellow. ORFs that are found in pTW2 and pTW3 and not in one of the six Xanthomonas strains are marked by the red triangles. Genes of the Xanthomonas strains that are not present in the insert sequences of pTW2 and pTW3 are indicated by the black rectangles. Numbers below the arrows indicate the ORF position. The predicted functions of the gene products from the six Xanthomonas strains and detailed comparisons with pTW2 and pTW3 are given in Tables S4 to S9 in the supplemental material. References were as follows: pTW2, EU333168 (the present study); pTW3, EU333169 (the present study); X. campestris pv. vesicatoria strain 85-10 (base 1118626 to base 1074579), AM039952; X. campestris pv. campestris strain ATCC 33913 (base 1040326 to base 995188), AE008922; X. campestris pv. campestris strain 8004 (base 4000216 to base 4045359), CP000050; X. axonopodis pv. citri strain 306 (base 1119791 to base 1071729), AE008923; X. oryzae pv. oryzae KACC 10331 (base 3859493 to base 3902413), AE013598; and X. oryzae pv. oryzae MAFF 311018, (base 3858820 to base 3899102), AP008229.

FIG. 2.

Alignment of the of the active-site regions of MprA and MprB with those of other metalloproteases belonging to the M4 family. The conserved HEXXH and GXXNEXXSD motif are shaded. Identical amino acid residues are indicated by bold letters. References were as follows: MprA and MprB (the present study); MprI from Pseudoalteromonas piscicida (34); EmpI from Pseudoalteromonas sp. strain A28 (27); vibriolysin from Vibrio vulnificus (6); and thermolysin from Bacillus thermoproteolyticus (CAA01492).

FIG. 3.

Domain structure of MprA (A) and MprB (B) and localization of the constructed derivatives of both proteases. For amino acid positions and similarities of domains and motifs, see Table 3. Abbreviations: SP, signal peptide; FTP, fungalysin/thermolysin propeptide motif; M4, peptidase M4 catalytic domain; M4_C, peptidase M4 alpha-helical domain; P_propr, proprotein convertase P domain.

FIG. 4.

Proteolytic activity of recombinant E. coli strains containing pTW2, pTW3, pMPRA, pMPRA.1, pMPRB, and pMPRB.1 to pMPRB.4 on skim milk-containing agar plates. The host for the plasmids pTW2 and pTW3 and the negative control pSKII⁺ was E. coli TOP10. The plasmids pMPRA, pMPRA.1, pMPRB, pMPRB.1 to pMPRB.4 and the negative control pET101/D were maintained in E. coli BL21. The recombinant E. coli strains were cultured on LB agar containing 2% skim. Halo formation indicated proteolytic activity. The gene regions cloned in pMPRA, pMPRA.1, pMPRB, and pMPRB.1 to pMPRB.4 are shown in Fig. 3.